Given the utility of murine models and the physiological and pathological significance of the aortic endothelium, we developed a simplified, nonenzymatic method for isolation and culture of murine aortic endothelial cells (MAECs). Aortic explants were initially cultured on fibronectin-coated plastic. Murine aortic endothelial cells migrated from the explants and proliferated. This expansion allowed for cultures to be established from the aortas of one or three mice. Murine aortic endothelial cells were then purified from expanded cultures by fluorescence-activated cell sorting for the uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate–labeled acetylated low-density lipoprotein. The majority of the cells in expanded cultures were as positive as human umbilical vein endothelial cells labeled in the same way. The most positive half of the labeled MAEC population was placed back in culture, and the cells formed “cobblestone” monolayers at confluence. Smooth muscle α-actin, which was present in aortic tissue and to a lesser extent in explant cultures before sorting, was not detected in selected MAECs. Western blotting and immunostaining also demonstrated the presence of the endothelial markers, platelet endothelial cell adhesion molecule-1, factor VIII–related antigen, and Bandeiraea simplicifolia lectin 1 binding. Murine aortic endothelial cells retained expected inflammatory functions: vascular cell adhesion molecule-1 protein was induced by bacterial endotoxin, and NO production was synergistically induced by the combination of endotoxin and interferon-γ. Our simple, efficient method will facilitate investigations of aortic endothelial cell function in vitro using murine models.